Limits...
Attenuation of loop-receptor interactions with pseudoknot formation.

Afonin KA, Lin YP, Calkins ER, Jaeger L - Nucleic Acids Res. (2011)

Bottom Line: Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others.Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor.Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA.

ABSTRACT
RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more 'G/C-rich' than their 'A/U-rich' natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for 'A/U-rich' GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

Show MeSH

Related in: MedlinePlus

Secondary structure diagrams and nomenclature of tectoRNA attenuators reported. (A) GAAA and GGAA molecular probes. (B) TectoRNA attenuators based on 11 nt receptor variants: their HD-forming module can assemble with the GAAA probe. (C) TectoRNA attenuators based on the R1 receptor motif and assembling with the GGAA probe. Indicated RNA constructs (labeled 1–17) are those with combinations of PK-forming modules and HD-forming modules that can form 3′PK (between nucleotides in blue) or 5′PK (between nucleotides in green). Red nucleotides are positions that vary from molecules 1 and 10 (with the classic 11 nt receptor and corresponding 3′ and 5′ PK-forming loops). Most constructs tested have a U at the pyrimidine position, which is labeled ‘Y’ within the HD-forming module. Additional constructs with combinations of PK-forming modules and HD-forming modules leading to mismatched PKs or with Y = C have been tested (see Figures 3 and 5 and Supplementary Table S1). Base-pairings are indicated according to Leontis and Westhof (43) annotation. The 11nt_GU receptor has been reported in previous studies as receptor C7.10 (8,15). For a description of the modularity of 11 nt receptor variants, see Supplementary Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3300017&req=5

gkr926-F2: Secondary structure diagrams and nomenclature of tectoRNA attenuators reported. (A) GAAA and GGAA molecular probes. (B) TectoRNA attenuators based on 11 nt receptor variants: their HD-forming module can assemble with the GAAA probe. (C) TectoRNA attenuators based on the R1 receptor motif and assembling with the GGAA probe. Indicated RNA constructs (labeled 1–17) are those with combinations of PK-forming modules and HD-forming modules that can form 3′PK (between nucleotides in blue) or 5′PK (between nucleotides in green). Red nucleotides are positions that vary from molecules 1 and 10 (with the classic 11 nt receptor and corresponding 3′ and 5′ PK-forming loops). Most constructs tested have a U at the pyrimidine position, which is labeled ‘Y’ within the HD-forming module. Additional constructs with combinations of PK-forming modules and HD-forming modules leading to mismatched PKs or with Y = C have been tested (see Figures 3 and 5 and Supplementary Table S1). Base-pairings are indicated according to Leontis and Westhof (43) annotation. The 11nt_GU receptor has been reported in previous studies as receptor C7.10 (8,15). For a description of the modularity of 11 nt receptor variants, see Supplementary Figure S2.

Mentions: 3D atomic models were manually constructed using the program Swiss-Pdb Viewer (35) following the RNA architectonics guidelines (24). All tectoRNA attenuators contain a heterodimer-forming module that assembles with a probe through two inter-molecular receptor/GNRA interactions (Figures 1 and 2). This module was modeled after the tectoRNA heterodimer (HD) (15,22,23) for which atomic model structures are presently available [PDB_ID: 2adt] (36,37). The 5′ and 3′ PK forming modules leading to the formation of 5′ and 3′ intra-molecular pseudoknots (PK), respectively, were modeled after the NMR structure of the Box H/ACA snoRNA bound to its rRNA target [PDB code: 2p89, 2pcv] (38,39) (Figure 1B and C and Supplementary Figure S1). In order to form the PK, the PK-forming module with a 10-bp apical stem, is linked through 4 nt to the HD-forming module, which includes a 3-bp stem (Figures 1 and 2). TectoRNA sequences (listed Supplementary Table S1) were checked for proper folding with the program Mfold (40,41) to maximize the stability of their secondary structure while minimizing the occurrence of alternative secondary structure folds. All tectoRNA attenuators are predicted to fold into a unique secondary structure prone to assemble with the probe. PKs with one or more GC bp are accurately predicted with Kinefold (42) (Supplementary Table S2).Figure 2.


Attenuation of loop-receptor interactions with pseudoknot formation.

Afonin KA, Lin YP, Calkins ER, Jaeger L - Nucleic Acids Res. (2011)

Secondary structure diagrams and nomenclature of tectoRNA attenuators reported. (A) GAAA and GGAA molecular probes. (B) TectoRNA attenuators based on 11 nt receptor variants: their HD-forming module can assemble with the GAAA probe. (C) TectoRNA attenuators based on the R1 receptor motif and assembling with the GGAA probe. Indicated RNA constructs (labeled 1–17) are those with combinations of PK-forming modules and HD-forming modules that can form 3′PK (between nucleotides in blue) or 5′PK (between nucleotides in green). Red nucleotides are positions that vary from molecules 1 and 10 (with the classic 11 nt receptor and corresponding 3′ and 5′ PK-forming loops). Most constructs tested have a U at the pyrimidine position, which is labeled ‘Y’ within the HD-forming module. Additional constructs with combinations of PK-forming modules and HD-forming modules leading to mismatched PKs or with Y = C have been tested (see Figures 3 and 5 and Supplementary Table S1). Base-pairings are indicated according to Leontis and Westhof (43) annotation. The 11nt_GU receptor has been reported in previous studies as receptor C7.10 (8,15). For a description of the modularity of 11 nt receptor variants, see Supplementary Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300017&req=5

gkr926-F2: Secondary structure diagrams and nomenclature of tectoRNA attenuators reported. (A) GAAA and GGAA molecular probes. (B) TectoRNA attenuators based on 11 nt receptor variants: their HD-forming module can assemble with the GAAA probe. (C) TectoRNA attenuators based on the R1 receptor motif and assembling with the GGAA probe. Indicated RNA constructs (labeled 1–17) are those with combinations of PK-forming modules and HD-forming modules that can form 3′PK (between nucleotides in blue) or 5′PK (between nucleotides in green). Red nucleotides are positions that vary from molecules 1 and 10 (with the classic 11 nt receptor and corresponding 3′ and 5′ PK-forming loops). Most constructs tested have a U at the pyrimidine position, which is labeled ‘Y’ within the HD-forming module. Additional constructs with combinations of PK-forming modules and HD-forming modules leading to mismatched PKs or with Y = C have been tested (see Figures 3 and 5 and Supplementary Table S1). Base-pairings are indicated according to Leontis and Westhof (43) annotation. The 11nt_GU receptor has been reported in previous studies as receptor C7.10 (8,15). For a description of the modularity of 11 nt receptor variants, see Supplementary Figure S2.
Mentions: 3D atomic models were manually constructed using the program Swiss-Pdb Viewer (35) following the RNA architectonics guidelines (24). All tectoRNA attenuators contain a heterodimer-forming module that assembles with a probe through two inter-molecular receptor/GNRA interactions (Figures 1 and 2). This module was modeled after the tectoRNA heterodimer (HD) (15,22,23) for which atomic model structures are presently available [PDB_ID: 2adt] (36,37). The 5′ and 3′ PK forming modules leading to the formation of 5′ and 3′ intra-molecular pseudoknots (PK), respectively, were modeled after the NMR structure of the Box H/ACA snoRNA bound to its rRNA target [PDB code: 2p89, 2pcv] (38,39) (Figure 1B and C and Supplementary Figure S1). In order to form the PK, the PK-forming module with a 10-bp apical stem, is linked through 4 nt to the HD-forming module, which includes a 3-bp stem (Figures 1 and 2). TectoRNA sequences (listed Supplementary Table S1) were checked for proper folding with the program Mfold (40,41) to maximize the stability of their secondary structure while minimizing the occurrence of alternative secondary structure folds. All tectoRNA attenuators are predicted to fold into a unique secondary structure prone to assemble with the probe. PKs with one or more GC bp are accurately predicted with Kinefold (42) (Supplementary Table S2).Figure 2.

Bottom Line: Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others.Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor.Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA.

ABSTRACT
RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more 'G/C-rich' than their 'A/U-rich' natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for 'A/U-rich' GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

Show MeSH
Related in: MedlinePlus